In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383.

Ufuk A, Somers G, Houston JB, Galetin A - Pharm. Res. (2015)

Bottom Line:
Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs.Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

Purpose: To assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383.

Methods: For all drugs, uptake at 5 μM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1-100 μM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated.

Results: Both cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.

Conclusions: This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

Fig2: Qualitative assessment of lysosomal sequestration of LysoTracker Red (LTR) and three drugs studied in NR8383 by confocal microscopy: (a) differential interference contrast image of a single NR8383 cell treated with 200 nM LTR; (b) the same cell being excited to detect LTR localised in lysosomes under control conditions; the localisation of LTR in the lysosomes of NR8383 was reduced in presence of 20 mM NH4Cl (c), 5 μM monensin (d), 10 μM nigericin (e), 5 μM clarithromycin (f) and 5 μM imipramine (g), and showed minor changes in presence of 5 μM formoterol (h).

Mentions:
The imaging of NR8383 cells was performed in order to confirm localisation of functional lysosomes in NR8383 and assess the effect of the chemical agents on LTR accumulation in these organelles. In addition, the lysosomal targeting of selected drugs was investigated. A differential contrast image was taken every time prior to excitation of the cells for LTR detection. A representative of this image is shown in Fig. 2a, demonstrating the presence of cytoplasmic vesicular structures likely to represent acidic organelles including lysosomes. The corresponding fluorescent image of the same cell is illustrated in Fig. 2b where LTR was localised in lysosomes, confirming the presence of these organelles in NR8383 cells. Treatment of NR8383 with LTR in the presence of NH4Cl (20 mM), monensin (5 μM) and nigericin (10 μM) showed reduced fluorescent signal up to 85% for all agents. The reduced accumulation of LTR in the presence of these agents (Fig. 2c, e, g) confirmed lysosomal targeting of this basic probe in NR8383. Assuming the competition of basic drugs for lysosomal sequestration, LTR was co-incubated with 5 μM of clarithromycin, imipramine and formoterol. Clarithromycin and imipramine decreased LTR accumulation by 86 and 72%, respectively (Fig. 2d, f), confirming indirectly the lysosomotropic properties of these two drugs. In contrast, the localisation of LTR was still evident in the presence of formoterol (Fig. 2h) which caused only 25% reduction in LTR accumulation, suggesting its marginal targeting of NR8383 lysosomes.Fig. 2

Fig2: Qualitative assessment of lysosomal sequestration of LysoTracker Red (LTR) and three drugs studied in NR8383 by confocal microscopy: (a) differential interference contrast image of a single NR8383 cell treated with 200 nM LTR; (b) the same cell being excited to detect LTR localised in lysosomes under control conditions; the localisation of LTR in the lysosomes of NR8383 was reduced in presence of 20 mM NH4Cl (c), 5 μM monensin (d), 10 μM nigericin (e), 5 μM clarithromycin (f) and 5 μM imipramine (g), and showed minor changes in presence of 5 μM formoterol (h).

Mentions:
The imaging of NR8383 cells was performed in order to confirm localisation of functional lysosomes in NR8383 and assess the effect of the chemical agents on LTR accumulation in these organelles. In addition, the lysosomal targeting of selected drugs was investigated. A differential contrast image was taken every time prior to excitation of the cells for LTR detection. A representative of this image is shown in Fig. 2a, demonstrating the presence of cytoplasmic vesicular structures likely to represent acidic organelles including lysosomes. The corresponding fluorescent image of the same cell is illustrated in Fig. 2b where LTR was localised in lysosomes, confirming the presence of these organelles in NR8383 cells. Treatment of NR8383 with LTR in the presence of NH4Cl (20 mM), monensin (5 μM) and nigericin (10 μM) showed reduced fluorescent signal up to 85% for all agents. The reduced accumulation of LTR in the presence of these agents (Fig. 2c, e, g) confirmed lysosomal targeting of this basic probe in NR8383. Assuming the competition of basic drugs for lysosomal sequestration, LTR was co-incubated with 5 μM of clarithromycin, imipramine and formoterol. Clarithromycin and imipramine decreased LTR accumulation by 86 and 72%, respectively (Fig. 2d, f), confirming indirectly the lysosomotropic properties of these two drugs. In contrast, the localisation of LTR was still evident in the presence of formoterol (Fig. 2h) which caused only 25% reduction in LTR accumulation, suggesting its marginal targeting of NR8383 lysosomes.Fig. 2

Bottom Line:
Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs.Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.

Purpose: To assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383.

Methods: For all drugs, uptake at 5 μM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1-100 μM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated.

Results: Both cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59-85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.

Conclusions: This study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.